Menu

Wednesday, March 4, 2015

Product Focus: Annexin V-FITC Kit

Benefits

Detects apoptosis earlier in the process than DNA-based assays (TUNEL).

Rapid labeling of cells. Cell staining takes only 10 minutes.

No cell fixation or processing required, reducing the detection time and allowing the cells to be used for further study.

PI is included with the kit to differentiate apoptotic cells from viable and necrotic cells.

Introduction

Annexin V-FITC kit provides a rapid and convenient assay for
apoptosis.Annexin V-FITC kit detects the externalization of
phosphatidylserine in apoptotic cells using recombinant annexin V
conjugated to green-fluorescent fluorescein isothiocyante (FITC) dye and
necrotic cells using propidium iodide (PI) by flow cytometry and
fluorescence microscope. The AnnexinV-FITC kit uses annexin V conjugated
with FITC to label phosphatidylserine sites on the membrane surface.
The kit also includes propidium iodide (PI) stains necrotic cells with
red fluorescence. After treatment with both probes, apoptotic cells show
green fluorescence, dead cells show red and green fluorescence, and
live cells show little or no fluorescence.

Catalogue

B32115 (50 rxns)

B32117 (100 rxns)

Annexin V-FITC

250 μl

250 μl×2

PI Staining Solution

250 μl

250 μl×2

1 × Binding Buffer

25 ml

25 ml×2

Storage

Store in refrigerator (2–8° C) and protect from light.

Experimental Protocol

1. Induce apoptosis in cells using the desired method. Prepare a
negative control by incubating cells in the absence of inducing agent.
2. Harvest the cells after the incubation period and wash in cold phosphate-buffered saline (PBS).
3. Re-centrifuge the washed cells, discard the supernatant, and resuspend the cells in 100 μl 1 × Binding Buffer.
4. Add 5 μL Annexin V -FITC and 5 μL PI Staining Solution to each 100 μL of cell suspension.
5. Incubate the cells at room temperature for 15 minutes.
6. After the incubation period, add 400 μL of 1 × Binding Buffer, mix gently and keep the samples on ice.
7. As soon as possible, analyze the stained cells by flow cytometry,
measuring the fluorescence emission at 530 nm (e.g., FL1) and >575 nm
(e.g., FL3). The population should separate into three groups: live
cells will show only a low level of fluorescence, apoptotic cells show
green fluorescence and dead cells show both red and green
fluorescence.Confirm the flow cytometry results by viewing the cells
under a fluorescence microscope, using filters appropriate for
fluorescein (FITC) and rhodamine (TRITC) or Texas RedR dye.

Stratech Scientific is a distributor of high quality, competitively
priced, reliable products for research laboratories throughout the UK
and Europe. Please contact us to find out which ranges we can supply in
your country.